Project Summary/Abstract Activation of nuclear receptors, a family of ligand-dependent transcription factors, is used extensively in pharmacology to develop drug targets for diverse medical conditions, including metabolic disease and cancer. While synthetic agonists and antagonists of nuclear receptors ameliorate symptoms of many diseases, clinical use of these compounds leads to frequent side effects that range in their severity. Type II receptors, such as FXR, LXR, and PPAR, which function in fatty acid, bile acid, cholesterol, and glucose metabolism, are permanently positioned in the nucleus regardless of the ligand status. The accepted paradigm regarding ligand activation of type II receptors is a two-step process: 1) the receptor is bound to DNA in complex with a co- repressor in absence of the ligand; 2) binding of the ligand induces a conformational change, co- repressor/co-activator exchange, and initiation of transcription. However, we observe that distribution of open chromatin sites changes upon ligand activation of nuclear receptors. In this project, I hypothesize that pioneer factor Foxa2 modulates chromatin accessibility by evicting nucleosomes to enable LXR? binding upon ligand activation, which challenges the accepted paradigm. Considering current limitations to develop highly selective synthetic ligands for nuclear receptors without significant side effects we propose that a genomic approach that separates LXR targets into distinct regulatory modes characterized by chromatin state and presence/absence of Foxa2 and LXR? binding sites will allow for genes with different physiological function to be targeted separately. We will test whether Foxa2 and LXR? binding is interdependent during ligand activation in Aim 1. We will ascertain the mechanism of Foxa2/LXR? interaction, discriminating between three possibilities in Aim 2. LXR could play the role of a co-activator without binding DNA, or both Foxa2 and LXR? need to be bound either in a proximal interaction or distal interaction. Successful completion of our project will change current thinking regarding activation of ligand-dependent gene expression and change the pharmacological approach to target nuclear receptors essential to metabolism.